Chromatography Columns, Systems and Methods

ABSTRACT

The present invention relates to axial flow chromatography columns, methods for separating one or more analytes in a liquid by the use of such columns, and systems employing such columns. The column comprises a first port and a second port, the first port and said second port being at essentially the same level or elevation above the level of the bed space on the chromatography column.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/459,525, allowed, which is a continuation of U.S. application Ser.No. 16/103,149 filed Aug. 14, 2018, abandoned, which is a continuationof U.S. application Ser. No. 15/379,449 filed Dec. 14, 2016, now U.S.Pat. No. 10,048,235, which is a continuation of U.S. application Ser.No. 14/252,874 filed Apr. 15, 2014, now U.S. Pat. No. 9,527,009, whichis a continuation of U.S. application Ser. No. 13/487,766 filed Jun. 4,2012, now U.S. Pat. No. 8,702,983, which is a continuation of U.S.application Ser. No. 11/879,126 filed Jul. 16, 2007, abandoned. Theentire contents of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to axial chromatography columns andmethods of separating one or more analytes present in a liquid from eachother using such chromatography columns.

BACKGROUND OF THE INVENTION

Chromatography is a well-established and valuable technique forseparating chemical and biological substances and is widely used inresearch and industry, finding many applications in compoundpreparation, purification and analysis. There are many different formsof chromatography, liquid chromatography being of particular importancein the pharmaceutical and biological industries for the preparation,purification and analysis of proteins, peptides and nucleic acids.

A typical liquid chromatography apparatus has an upright housing inwhich a bed of packing material, which is usually particulate in natureand consists of a porous medium, rests against a permeable retaininglayer. A liquid mobile phase enters through an inlet, for example at thetop of the column, usually through a porous, perforated filter, mesh orfrit, moves through the bed of packing material and is removed via anoutlet, typically through a second filter, mesh or frit.

Columns used in liquid chromatography typically comprise a tubular bodyenclosing the porous chromatography medium through which the carrierliquid or mobile phase flows, with separation of substances or analytestaking place between the mobile phase and solid phase of the porousmedium. Typically, the porous medium is enclosed in the column as apacked bed, generally formed by consolidating a suspension of discreteparticles, known as slurry that is pumped, poured or sucked into thecolumn, usually from a central bore or nozzle located at one end of thecolumn. The production of a stable, even bed is critical to the finalseparation process and optimum results are found using bores which arecentrally positioned through the column end. Systems for producing suchstable, even beds are known in the art and include, for example,compressing the bed by means of an end unit.

Following column packing and prior to use it is necessary to install thecolumn by connecting it to a chromatography system, usually comprising apump, detectors and inlet and outlet manifolds. When installing thecolumn, it is essential to avoid any draining of the column from liquidas well as to avoid introduction of air into the column and the packedbed, respectively. Furthermore, it may be necessary to purge the systememploying the column of any air upstream of the column. Typically, priorart columns and systems accomplish column installation and/or purging orventing by means of a valve (a “vent” or “purge” valve) at one end ofthe column, usually at its base.

In practice, the valve is used together with the column, which meansthat the column is connected and disconnected from the system upstreamof the valve. For a disposable column, the introduction of such anadditional valve is costly. By virtue of its position, it increases boththe risk of siphoning of liquid from the column and also the likelihoodof further air being introduced into the column. Furthermore, theposition of the valve imposes constraints on locating the column withinthe laboratory and can cause problems in terms of user accessibility,since access to both the top and bottom of the column is required.

JP 63293456 (Sekisui Chemicals Co.) describes a column holder to supportcolumns having been prepared with a stationary phase and being equippedwith a discharge aperture at the base and a ventilation device at thetop. The column holder is designed such that on the application ofgravity or a centrifugal force, to facilitate chromatographic separationof materials on the stationary phase, any surplus fluid remaining abovethe stationary phase can be removed without allowing air to enter thestationary phase within the column. The discharge aperture is connectedto a conduit which is branched at a position that is at the same or ahigher level than the upper end of the stationary phase solution and hasthree open ends, a first at the connection point with the dischargeaperture, a second at a position above the surface level of the solutionand a third located below the level of the discharge aperture. Theconduit is configured such that any surplus fluid can be removed fromthe column through the third open end where it is collected in anacceptor vessel.

Despite the high level of activity in the field of chromatography overmany years there is still a need for a simple axial column that obviatesthe need for a vent valve or complex column holder and reduces the riskof air entering the column and/or liquid siphoning from it. To date, noaxial chromatography columns are available which meet this need and/orprovide improved user accessibility.

Definitions

“Analyte” shall be defined as a substance, compound or chemical ofnatural or synthetic origin, or a reaction product or derivative ormetabolite thereof. For the avoidance of doubt, the term shall includebiological molecules, such as proteins, peptides, amino acids andnucleic acids, and synthetic molecules such as dmgs and/or pro-dmgs.

“Distribution channel” refers to structures through which fluids areintroduced to an enclosure or bed space for a packed bed ofchromatography medium from a cross-sectional zone.

A disposable column is characterized by a pre-treatment of thechromatography medium in order to reduce installation and qualificationwork otherwise required with non-disposable columns. As a minimum, thepre-treatment involves the formation of the bed of porous medium.Additional pre-treatment can be reduction of microbiological burden,sterilization, depyrogenation etc.

Disposable column may be used as single-use columns, which means thatthe user is not performing cleaning regimes that require qualification(e.g. testing, validation, etc) of the packed bed before repeated use.

One embodiment of a disposable column is a complete column that isdelivered prepacked with chromatography medium.

Another embodiment of a disposable column consists of a first devicerepresenting a frame or vessel designed to resist pressure and loadexerted on one or multiple lateral surfaces of the packed bed duringoperation in order to provide dimensional stability for a packed bed,and a second device, representing a container, shell, cartridge, bag orthe like containing the porous medium or bed that is attached to thefirst device for operation. With the latter embodiment, the porousmedium is contained in the secondary container and can be replaced whilethe frame is re-usable. In this case, the degree of compression of theporous medium required for operation may be adjusted after inserting thecontained medium into the frame (see, for example US2002/0166816, Allen& Dawson and WO2005/009585, Sigma-Aldrich Co.).

“Level” shall be defined as a horizontal plane or specified height.

SUMMARY OF THE INVENTION

The object of the invention is to provide a chromatography column whichovercomes the drawbacks of the prior art systems. This is achieved bythe chromatography column as defined in claim 1.

One advantage with such a chromatography column is that it reduces therisk of siphoning from the column.

Another advantage of the invention is that it is easy and cheap toproduce, compared with existing columns that required an adjacent purgevalve. This is especially important when using the column as adisposable column.

A further advantage of the chromatography column is that it is easier touse than conventional columns because both the first port and secondport which provide an inlet and an outlet for liquids such as mobilephase are at essentially the same level or elevation above the level ofthe bed space on one end of the column and thus provide improved useraccessibility.

The simplicity of the design comprising few interconnected elements isadvantageous in that it reduces the requirement for dynamic sealstightening and moving or rotating parts, as required for a (disposable)purge valve, hence decreasing the likelihood of leakage or contaminationdue to sanitary problems. These features are especially important fordisposable columns comprising a reduction of microbiological burdenduring preparation (production) of the column, as well as to maintainthose conditions during storage and shipping of the column.

Still a further advantage is that the chromatography column is scalablein that increasing or decreasing the column size leads to a predictableperformance.

According to a first aspect of the invention, there is provided an axialflow chromatography column comprising:

-   -   a housing comprising a side wall;    -   opposed, axially spaced first and second end units separated by        the side wall; a first filter which is adjacent to the first end        unit and a second filter which is adjacent to the second end        unit wherein the filters together with the side wall define an        enclosed bed space for containing a bed of particulate medium        therein;    -   the first end unit comprising a first port which is in fluid        communication with the enclosed bed space for adding a liquid to        or removing a liquid from the bed space; the second end unit        comprising a second port which is in fluid communication with        the enclosed bed space for adding a liquid to or removing a        liquid from the bed space; and characterised in that the first        port and the second port are at essentially the same level or        elevation above the level of the bed space on the chromatography        column.

In a second aspect, there is provided a method for separating one ormore analytes in a liquid from each other, comprising applying theliquid containing the one or more analytes to an axial chromatographycolumn as hereinbefore described, the column containing a bed ofparticulate medium therein, eluting the one or more analytes with amobile phase, and collecting fractions of the mobile phase eluting fromthe column.

In a third aspect of the invention, there is provided a system forseparating one or more analytes in a liquid from each other, the systemcomprising: an inlet or inlet manifold in fluid communication with theliquid;

-   -   a pump;    -   a chromatography column as hereinbefore described; and an outlet        or outlet manifold.

Embodiments of the invention are defined in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic transverse sectional view of a chromatographycolumn of the prior art showing the basic features thereof.

FIG. 2a , FIG. 2b and FIG. 2c are simplified schematic diagramsillustrating prior art (FIGS. 2a and 2b ) columns and a column inaccordance with the invention (FIG. 2c ).

FIG. 3 is a transverse section of a column in accordance with theinvention.

FIG. 4 is a three-dimensional schematic of a chromatography columnaccording to the invention.

FIG. 5 a is a schematic diagram of a system using a column known in theprior art.

FIG. 5b is a schematic diagram of a system employing a column inaccordance with the invention.

FIG. 6 is a chromatogram showing the chromatographic separation ofacetone on a chromatography column according to the invention, both inupflow (dotted line) and downflow (solid line) mode.

FIG. 7 describes a method for calculating the reduced plate height andasymmetry factor from an eluted peak.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows schematically the general components of a chromatographycolumn 1 as known from the prior art (for example, see FTS 6,524,484).The column has a cylindrical fluid-impermeable side wall 11, e.g. ofstainless steel or a high-strength/reinforced polymeric material whichmay be translucent. The open top and bottom ends of the side wall 11 areclosed by top and bottom end assemblies or units 12, 13. Each end unithas a fluid-impermeable end plate 3 fitting sealingly to plug theopening of the cylindrical wall 11, and preferably made of stainlesssteel or high-strength engineering plastics material, e. gpolypropylene. The end plates are backed up by metal retaining plates 2bearing against their outer surfaces and projecting radially beyond theside wall as retaining flanges 22 through which adjustable tension rods14 are secured. These link the top and end assemblies 12, 13 and helpthe construction to withstand high fluid pressures.

Each end plate 3 has a central through-opening 31 for communicationbetween the exterior of the column and the packing bed space 9 definedby the side wall 11 and end assemblies 12, 13. Access through theopening 31 is subdivided into separate conduits, connected externallythrough a connection manifold 8.

A filter layer 4, typically of filtered or woven plastics or steel,extends across the area of the bed space 9 at the inner surface of theend plate 3. The inner surface 35 of the end plate 3 is recessed behindthe filter layer 4, e.g. conically as illustrated, and preferably withthe use of support ribs (not indicated) supporting the filter layer 4from behind, to define between them a distribution channel 34. One ofthe communication conduits, a mobile phase conduit 33, opens inwardlyinto this distribution channel 34, as well as outwardly to a mobilephase connector 81 of the manifold 8.

From the manifold 8, an access valve device 5 projects inwardly throughthe end plate opening 31 and sealingly through a central orifice 41 ofthe filter layer 4. The access valve 5, governs the communication of oneor more conduits from the manifold 8 directly to the bed space 9, i.e.bypassing the filter layer 4. Indicated here are first and second valvedconduits 51, 61 governed by the valve 5, and connected externallythrough connectors 82 of the manifold 8.

In a typical operation of the column, a packed bed of particulatestationary phase material fills the bed space 9 between the top andbottom filter layers 4. The valve devices 5 being closed, a mobile phaseis fed in through mobile phase connector 81 (arrow “A”), passes throughconduit 33 into the distribution channel 34 and through the filter layer4 to elute down through the packed bed, effecting separation of itscomponents or analytes. Liquid eluate passes thought the filter layer 4of the bottom end assembly 13 and out through the mobile phase connector81 thereof (arrow “B”) for collection as appropriate. While this is anexample of “downflow” chromatography, in that chromatographic separationis effected by the downward movement of the mobile phase through thecolumn, the skilled person will understand that separation mayalternatively be achieved by “upflow” chromatography, simply by pumpingmobile phase upwards through the column and thus reversing the directionof flow. In this mode, mobile phase would enter the column at connector81 (arrow “B”), move upwards through the stationary phase or particulatemedium, and be collected from connector 81 (arrow “A”) at the top of thecolumn.

When installing the column, it is essential to avoid any draining of thecolumn from liquid as well as to avoid introduction of air into thecolumn and the packed bed, respectively. Furthermore, it may benecessary to purge the system employing the column of any air upstreamof the column. This is achieved by means of a vent valve 5 which islocated at the bottom of the column.

FIG. 1 and the above explanation are to illustrate general relationshipsof components and a typical mode of operation. The skilled person willunderstand, and it will also appear from the following description, thatother specific constructions and modes of operation may be appropriatefor different kinds of process.

FIGS. 2a and 2b show simplified and schematic representations of theconfiguration of a prior art column. The column 101 has a first port 133and a second port 140 for the introduction or collection of mobilephase. After installation of the column and prior to use, air needs tobe removed from the column by means of venting/purging. This may beachieved by means of a vent valve 105 with a vent outlet which is eitheran integral part of the column 101 (as shown in FIG. 2b ) or isconnected to it at a point beyond the second port 140 (not shown in FIG.2a ).

FIG. 2c is a simplified and schematic representation of a configurationof a column 101 in accordance with the invention. The first port 133,which is intended for the introduction or collection of mobile phase, islocated at the top of the column 101 (as with columns of the prior art).The second port 140, which is in fluid communication with the bottom ofthe column, is at essentially the same level or elevation as the firstport 133. This is achieved by means of a hollow member 160 which allowsfluid such as air or mobile phase to flow between the bottom of thecolumn 101 and second port 140. This configuration eliminates the needfor a vent valve and reduces the risk of both siphoning from the columnand introducing air into the column.

FIG. 3 shows a transverse sectional view of a column in accordance withthe invention. The column 201 comprises a tubular housing 211, a firstend unit 212 (partially shown) and a second end unit 213, securedtogether to form a fluid tight seal by means of tension rods 214 withheads 216. First filter 204 and second filter 206 are adjacent to thefirst end unit 212 and second end unit 213, respectively. These filters204, 206, together with side wall 211, define a bed space 209 forcontaining a bed of particulate medium.

The housing 211 and end units 212, 213 are typically composed ofstainless steel or a high-strength plastic material such aspolypropylene. In a preferred embodiment, where the column is to be usedfor the separation of biologically active substances, the material isbiologically inert such that it does not elicit an immune response inhumans in accordance with United States Pharmacopia (PTSP) <88> classVI. Tension rods 214, with heads 216, secure the end units 212, 213 tothe housing 211 to form a fluid-tight bed space 209 which is capable ofwithstanding high operating pressures.

Valve means 220 and first port 233 are shown in the figure. The secondport 240 comprises a passageway 242 which extends through second endunit 213 to, and is in fluid communication with (via hollow member 260),bed space 209 from which liquid can be added or collected. As is evidentfrom the figure, the second port 240 is at essentially the same level orelevation as the first port 233, thus facilitating the addition andcollection of mobile phase to/from the column. This arrangement hasfurther advantages in that it assists in the installation of the column,decreases the risk of syphoning and reduces the likelihood ofintroduction of air into the column.

The column can be packed with particulate medium in the form of a slurrythrough valve means 220, the valve means 220 comprising a central bore221 and nozzle 224. A bed of packed particulate medium is obtained byconventional means well known in the art, for example by the movement ofone of the end units to compress the bed. In FIG. 3 the nozzle 224 isshown in its retracted position but it will be understood that it can bemoved to a position within the bed space 209 to facilitate filling ofthe column. A wide range of nozzles can be used which facilitate thedistribution and even packing of slurry within the bed space. Onealternative for achieving an open/closed functionality at the packingvalve and nozzle respectively is to have a nozzle that is fixed in thebed space (and thus not retractable) and located adjacent to a moveableelement or sleeve on the inside or outside of the nozzle that opensand/or closes the nozzle depending upon its position. Filters 204, 206are each positioned on the interior face of the end units 212, 213 andact to define the bed space (together with side wall 211) and to preventleakage of particulate medium from the bed space 209.

Mobile phase or liquid containing one or more analytes or substances forseparation on the column is added via first port 233. The liquid thenpasses through the filter 204 into the bed space 209 that is packed withparticulate medium (not shown). Chromatographic separation of analyte(s)which has been introduced onto the particulate medium in this manner iseffected by introduction of, and elution by, mobile phase. The mobilephase will finally exit the column through second filter 206 and viapassageway 242 to second port 240. The resulting fractions of mobilephase, which contain different analytes, can then be collected.

It will be understood by the skilled person that the column may beoperated in either a “downflow” mode, as described above, or in an“upflow” mode where the direction of flow of the mobile phase isreversed such that it moves up the column. In upflow mode, mobile phasewill enter the column via second port 240, move along passageway 242 andupwards through the bed of particulate medium in bed space 209, to exitthe column for collection at first port 233.

In the embodiment shown, hollow member 260 is an integral part of thecolumn. However, it will be understood that by means of connectors andappropriate tubing made from a suitable material (e.g. polypropylene,polyurethane, etc.) that the hollow member 260 need not be integral tothe column.

The application and collection of mobile phase at the same elevation ona single end unit simplifies use, in terms of operator access andhandling, reduces the risk of air accessing the system and decreases thespace necessary to set up the column.

The embodiment shown in FIG. 3 comprises a valve means (220) for theintroduction and/or removal of particulate medium from the column. Itwill be understood that such a valve is not an essential feature of theclaimed invention as some columns (e.g. pre-packed, disposable columns)may not require the addition or removal of particulate medium to beperformed by the end user or are prepared (packed) with a differenttechnique not requiring the use of such a valve means.

FIG. 4 presents a three dimensional schematic representation of thecolumn of FIG. 3, from which the external features of the column areevident. The column comprises a first end unit 312, second end unit 313and housing 311 which are secured together to form a fluid-tight seal bytension rods 314 and heads 316. Particulate medium in the form of aslurry can be introduced into the bed space (not shown) via valve means320. First port 333 serves as a conduit for mobile phase or liquidcontaining analyte(s) to be separated on the particulate medium. Hollowmember 360, which is in fluid communication with the bed space via anoutlet at the base of the column (not shown), ends in second port 340from which appropriate fractions of mobile phase eluted from the columnmay be collected. As can be seen, second port 340 is at essentially thesame level or elevation as the first port 333 through which mobile phasecan be introduced (or collected). This arrangement facilitates useroperation and sample handling. In the embodiment shown in FIG. 4, thecapacity of the column is approximately 10 liters; it will be understoodthat a wide range of column capacities is possible, typically rangingfrom 0.1 to 2000 liters. Preferred capacities when using the column as adisposable column are in the range of 0.5 to 50 liters.

FIGS. 5a and 5b schematically compare a system incorporating a prior artcolumn (FIG. 5a ) having an integrated vent valve to a system using acolumn in accordance with the invention (FIG. 5b ). The vent valve shownin FIG. 5a is a rotary valve type, but it will be understood that othervalve principles (pinch valves, membrane valves, etc) may also beemployed to achieve the vent valve functionality.

Following installation of the prior art system (FIG. 5 a), air must beremoved from the system by priming it. The system comprises an inletmanifold 408, pump 470, sensors 472-476, column valve 407, outletmanifold 409 and column 401 (the dotted rectangle shown enclosing thecolumn 401 and vent/purge valve 405 indicates that the column and thevent valve are used as a combined unit such that the vent valve isattached to the packed and primed column when installing the column in achromatography system). As described above, the purpose of the ventvalve is to protect the column from draining and/or the introduction ofair when installing it in a system or when removing/disconnecting thecolumn from a system. The column valve 407 controls connection of thecolumn 401 to the inlet 408 and/or outlet 409 manifolds and thus governswhether the column 401 is “offline” or “online”. In FIGS. 5a and 5b ,the column valve is of a rotary valve type, but it will be understoodthat other valve principles (e.g. pinch valves, membrane valves, etc)may also be employed to achieve the column valve functionality. Fluidconnectivity with the column 401 and inlet 408/outlet 409 manifolds iscontrolled by means of several gateways within the valve 407 asindicated by positions 1-4 in the diagram). When valve 407 makesconnection between positions 3 and 4 and positions 1 and 2, the columnis bypassed. When the rotary valve is turned by 90 degrees, connectionbetween positions 3 and 1 and in between positions 4 and 2 is made,which means that the column is inline or online and connected in upflowmode/flow direction. As explained above, other valve principles (e.g.pinch valves, membrane valves, etc) and a wide range of other valveconfigurations (upflow and/or downflow modes as well as connection ofmultiple columns) may be employed to achieve the column valvefunctionality.

Air is removed from the system by means of vent/purge valve 405 whichallows priming of the system, in particular priming of conduit 480, andpurging of any air within it.

The system shown in FIG. 5a is intended to be used in an upflow mode;thus liquid from inlet manifold 408 enters column 401 via conduit 480 atsecond port 440 and moves upwards through the packed bed (not shown)exiting at first port 420. Liquid (e.g. mobile phase or samplecontaining analytes to be separated on the column) is taken up frominlet manifold 408 and transferred to the column 401 under pressure bymeans of pump 470 via column valve 407. Sensors 472-476 can be used tomeasure environmental, physical and chemical conditions in the system(e.g. pressure, flow, conductivity, temperature, pH, LTV absorbance, airetc). These sensors can be used to control the operation of the column,for example by regulating flow rates of mobile phase through the column.Liquid emerging from the column from first port 420 is transferred viacolumn valve 407 to outlet manifold 409 for collection.

FIG. 5b exemplifies a system using a column in accordance with theinvention. The component parts are the same as described above for FIG.5 a except that there is no vent/purge valve 405. In this configurationthe level or elevation of the first port 420 and the second port 440above the level of the bed space (not shown) in the column isessentially the same, the second port 440 being connected to the base ofthe column 401 by means of hollow member 460. While hollow member 460 ispart of the column in accordance with the invention, the correspondingliquid conduit 480 in FIG. 5a , which shows the prior art configuration,is part of the chromatography system. The column according to theinvention with its hollow member 460 is already purged of air and may beready for use when installed in the system. Especially for disposable,ready-to-use columns, the invention avoids the need for a disposablepurge valve delivered with each individual column, which significantlyreduces cost.

Following installation, the system is purged upstream of the columnvalve 407 when the column 401 is bypassed (i.e. the column is“offline”). When switching the column “inline”, only a negligible volumeof air will remain between the column valve 407 and conduit 480.

The system shown in FIG. 5b can then be used in essentially the samemanner as described above for the prior art system. In upflow mode,liquid will be aspirated from manifold 408 by pump 470 and directed, viavalve 407, into second port 440 of column 401. The liquid will then moveup the column through a bed of particulate medium (not shown) to exit atfirst port 420 and be directed, by column valve 407, to outlet manifold409 for collection. Sensors 472-476 can be used to monitorenvironmental, physical and chemical conditions within the system andthus to regulate its operation.

FIG. 6 shows the chromatographic separation efficiency by example of atracer pulse experiment achieved on a 10 litre column in accordance withthe invention, operated in both downflow (solid line) and upflow (dottedline) mode. The column was packed with a bed of CAPTO™ Q anion exchangeresin (GE Healthcare, Uppsala, Sweden) of 85 pm agarose particlediameter. The column had a volume of 10.8 l, a diameter of 263 mm and abed height of 200 mm. Acetone (1% of packed bed volume) was used as atracer substance and eluted from the column using water as mobile phaseand the absorbance monitored at 280 nm. As can be seen from Table 1below, excellent column efficiency was observed with the 85 pm agarosemedium used, either in downflow (solid line) or upflow (dotted line)mode.

The data shown in Table 1 and FIG. 6 were obtained using achromatography column in accordance with the invention which furthercomprises a transverse distribution channel and wherein the outlet ofthe first port and the transverse distribution channel have anasymmetric configuration. A chromatography column having such anarrangement is the subject of the Applicant's (GE HealthcareBio-Sciences AB) copending patent application entitled “AxialChromatography Columns and Methods” filed on the same day herewith as GB0614316.8.

TABLE 1 Observed Acceptance Plates/meter (N/m) 4430 >3700 (for 85 pm)Reduced plate height (h) 2.5 <3.0 Peak asymmetry (Af) 1.14 0.8-1.8The data from Table 1 were derived from the chromatogram of FIG. 6 asdescribed below.

As a measure for column efficiency, the reduced plate height isdetermined with help of the peak width w_(h) at half the height of theeluted peak, as shown in FIG. 7. This procedure is an approximationvalid for the gaussian-shaped. In practice, eluted peaks often deviatefrom this ideal gaussian shape and peak skewness is describedqualitatively by a so-called asymmetry factor A_(f), where ‘leading’ inthe RTD is indicated by A_(f)<1 and ‘tailing’ by A_(f)>1. Commonlyapplied acceptance criteria for the asymmetry factor are0.8<A_(f)<1.5-1.8, depending on the type of application.

$h = {\frac{HETP}{d_{p}} = {\frac{L}{d_{p}}\frac{1}{5.54}\left( \frac{w_{h}}{V_{R}} \right)^{2}}}$A_(f) = b/a

(see FIG. 7)

As a rule of thumb, the characteristic dispersion of the mediumtypically gives a reduced plate height in the range h=1.5-2.0 at anoptimized superficial velocity when considering the highly porous mediumused for protein chromatography in biotechnological downstreamprocessing. The ideal efficiency of the medium has to be compared to theexperimentally determined efficiency of the chromatographic system,where an increase in the reduced plate height is a result of additionaldispersion from peripherals, sample volume, bed heterogeneities anddistribution system. In practice, a typical standard installationqualification of a chromatographic unit used in ion exchange separationsof proteins is an experimentally determined reduced plate height ofh_(Unit,Apparent)<3.0.

A_(f) asymmetry factor

d_(p) particle diameter

h reduced plate height

HETP height equivalent of a theoretical plate

L bed height, packed bed

u_(s) superficial velocity in packed bed

V_(R) retention volume

W_(h) peak width at 50% of max. peak height

It is apparent that many modifications and variations of the inventionas hereinabove set forth may be made without departing from the spiritand scope thereof. The specific embodiments described are given by wayof example only, and the invention is limited only by the terms of theappended claims.

What is claimed is:
 1. An axial flow chromatography column comprising: ahousing comprising a side wall; opposed, axially spaced first and secondend units separated by said side wall; the first end unit comprising afirst port which is in fluid communication with said enclosed bed spacefor adding a liquid to or removing a liquid from the bed space; thesecond end unit comprising a second port which is in fluid communicationwith the enclosed bed space for adding a liquid to or removing a liquidfrom the bed space; and wherein said first port and said second port aredisposed at or above the level of the bed space on said chromatographycolumn.
 2. The chromatography column according to claim 1, wherein thesecond port is in fluid communication with the bed space by means of ahollow member connected to a passageway in the second unit.
 3. Thechromatography column according to claim 2, wherein said hollow memberis an integral part of the column.
 4. The chromatography columnaccording to claim 2, wherein said passageway extends from the bed spacethrough a lateral wall of the second end unit.
 5. The chromatographycolumn according to claim 1, wherein the first end unit additionallycomprises a valve which is in fluid communication with the enclosed bedspace, the valve being operably openable and closable to allow filing ofthe bed space with the particulate medium through said passageway. 6.The chromatography column according to claim 5, wherein a longitudinalmember of said valve comprises a nozzle.
 7. The chromatography columnaccording to claim 6, wherein said nozzle is fixed within the bed spaceor retractable to a position outwith the bed space.
 8. Thechromatography column according to claim 5, wherein said valve does notallow emptying of the bed space of particulate medium.
 9. Thechromatography column according to claim 1, wherein said column ispre-packed with particulate medium.
 10. The chromatography columnaccording to claim 9, wherein said column is a disposable column. 11.The chromatography column according to claim 1, further comprising afirst filter which is adjacent to said first end unit and a secondfilter which is adjacent to said second end unit wherein said filterstogether with the side wall define an enclosed bed space for containinga bed of particulate medium therein.
 12. The chromatography columnaccording to claim 1, further comprising at least one distributionchannel therein.
 13. The chromatography column according to claim 12,wherein said at least one distribution channel comprises a transversedistribution channel.
 14. The chromatography column according to claim1, wherein at least one of the housing, the first end unit, the secondend unit, the first port and/or the second port comprise stainless steelor a high-strength/reinforced polymeric material.
 15. The chromatographycolumn according to claim 14, wherein the polymeric material comprisespolypropylene.
 16. A method for separating one or more analytes in aliquid from each other, comprising applying said liquid containing saidone or more analytes to an axial chromatography column according toclaim 1, said column containing a bed of particulate medium therein,eluting said one or more analytes with a mobile phase, and collectingfractions of said mobile phase eluting from the column.
 17. A system forseparating one or more analytes in a liquid from each other, said systemcomprising: an inlet or inlet manifold in fluid communication with saidliquid; a pump; a chromatography column according to claim 1; and anoutlet or outlet manifold.
 18. A system according to claim 17,additionally comprising a valve which is operably openable and closableto allow the addition of liquid to, or the removal of liquid from, thecolumn bed space.